The invention concerns methods for the efficient synthesis of oligoketide thioesters, including diketide and triketides, which are useful as intermediates in polyketide production and to methods to use these intermediates. The methods of synthesis are suitable for liquid phase as well as solid-phase combinatorial synthesis. The invention also includes polyketide and tailored polyketide products.
The creation of novel macrolide polyketides has been achieved through genetic manipulation of polyketide synthases. The modular nature of the Type 1 polyketide synthases allows for domain exchange between different polyketide synthase genes, resulting in hybrid genes which produce polyketide synthases with altered properties that result, in turn, in modified macrolide structures. Thus, it is possible to control chain length, choice of chain extender unit, degree of xcex2-carbon oxidation level, and to some degree stereochemistry. The choice of starter unit has been more difficult to control. Two complementary approaches have been described.
Dutton, et al., J. Antibiotics (1991) 44:357-365 demonstrated that the avermectin polyketide synthase was somewhat flexible in choice of starter units. When denied the natural starter unit through inactivation of the branched-chain amino acid dehydrogenase, the avermectin polyketide synthase will accept a variety of xcex1-branched carboxylic acids as the starter unit. However, only about 30 acids out of nearly 800 candidate acids tried were accepted. Acids without an xcex1-branch appear to be metabolized through xcex2-oxidation until an xcex1-branch is reached, further limiting this methodology. Marsden, et al., Science (1998) 79:199-202 exchanged the native loading domain of the erythromycin PKS with that from the avermectin polyketide synthase, resulting in a hybrid PKS having the same loosened starter unit specificity as the avermectin PKS. Clearly, the native specificities of enzymatic domains will always be a limitation on the flexibility of resulting hybrid systems.
A more general method for controlling starter unit specificity has been described by Jacobsen, et al., Science (1997) 277:367-369. Inactivation of the ketosynthase in module 1 (KS1) of the erythromycin PKS (DEBS) results in an enzyme (KS1xc2x0-DEBS) incapable of initiating polyketide synthesis using precursors normally available to the cell. When supplied with a suitable thioester of the diketide product of module 1 or its analogs, however, KS1xc2x0-DEBS efficiently incorporates these into full-length polyketides. Subsequent experiments have demonstrated that a very wide range of diketide analogs are accepted by KS1xc2x0-DEBS, making this a very general method for production of analogs of the polyketide precursor of erythromycin, 6-deoxyerythronolide B (6-dEB), with variations at the positions controlled by the starter unit. Further, this method allows for production of 6-dEB analogs altered at the 12-position; this is equivalent to altering the substrate specificity of the module 1 acyltransferase (AT1) which transfers the first extender unit. While this has been accomplished through the above described domain exchange experiments as well, the xe2x80x9cdiketide methodxe2x80x9d allows for introduction of 12-position substituents which are not available from nature. Furthermore, triketide analogs are accepted, opening the 10- and 11-positions of 6-dEB for modification. The 6-dEB analogs obtained can be further converted into analogs of erythromycin by feeding to a suitable converter strain, such as a strain of Saccharopolyspora erythraea containing a non-functional erythromycin PKS. The resulting erythromycins have altered side-chains at the 13-position as well as other optional modifications, and show altered biological activity. These erythromycin analogs can also be produced by introducing the KS1xc2x0-mutation into an erythromycin-producing strain of Saccharopolyspora erythraea, then supplying the mutant strain with suitable diketide or triketide thioesters as described above.
Implementation of this method requires the availability of the N-acylcysteamine oligoketide thioesters. Synthetic methods available in the art for these thioesters do not lend themselves to efficient, economical synthesis, or to the systematic production of variants. The diketide and triketides also typically contain chiral centers requiring the control of absolute or relative stereochemistry.
Cane, D. E., et al., J Am Chem Soc (1987) 109:1255-1257 describes a three-step process to produce the N-acetylcysteamine thioester of (2S,3R)-2-methyl-3-hydroxy pentanoic acid. The method relies on the use of a chiral reagent N-propionyl-(4S)-4-isopropyl-2-oxazolidinone for control of the absolute stereochemistry of the product: 
This material results from the acylation of (4S)-4-isopropyl-2-oxazolidinone with propionyl chloride, typically using a strong base such as n-butyllithium at low temperature. The Cane process is an aldol condensation of this starting material with propionaldehyde in the presence of dibutylboron triflate (Bu2BOTf), followed by hydrolysis of the imide (lithium hydroperoxide) and thioesterification with N-acetylcysteamine in the presence of diphenyl phosphorylazide and triethylamine. This multi-step process is inefficient, with substantial losses accompanying the hydrolysis step.
Cane, D. E., et al., J Antibiotics (1995) 48:647-651 was able to improve yields using a five-step process which replaces the aldol condensation with a Claisen condensation between the lithium enolate of the propionyl oxazolidinone (N-propionyl-(4S)-4-benzyl-2-oxazolidinone was used as the stereochemistry controlling starting material in this method) and propionyl chloride followed by reduction of the resulting xcex2-ketoester product using zinc borohydride. Protection of the xcex2-hydroxy substituent as a tert-butyldimethylsilyl ether preceded hydrolysis of the imide, which again required lithium hydroperoxide. The protecting group gave improved yields from hydrolysis, but required an additional two steps to add and remove. This longer process also suffers from the use of zinc borohydride, which is not commercially available.
Cleavage of the N-acyloxazolidinones resulting from either aldol or Claisen condensations as described above is problematic. Various methods of cleavage are known in the art, including that of Evans, D. A., et al., Tetrahedron Lett (1987) 28:6141, in which undesired reaction at the oxazolidinone carbonyl during hydrolysis is suppressed by the use of lithium hydroperoxide. This process requires the use of concentrated solutions of hydrogen peroxide, which are explosive and dangerous for large-scale processes. The N-acyloxazolidinones are unreactive towards thiols or thiolates, although some conversion to thioesters can be observed using concentrated solutions of lithium thiolates in tetrahydrofuran. The low solubility of the thiolates in tetrahydrofuran combined with epimerization of chiral diketides due to the basicity of the thiolates limits the utility ofthis method. Miyata, O., et al., Syn Lett (1994) 637-638, describes conversion of N-acyloxazolidinones to S-benzylthioesters through the use of lithium benzylthiotrimethylaluminate. The production of more complex thioesters containing groups capable of binding Lewis acids like trimethylaluminum, such as those based on N-acylcysteamine, has not been reported.
The N-acetylcysteamine thioesters of larger oligoketides have also been prepared. Cane, D. E., et al., J Am Chem Soc (1993) 115:522-526 synthesized the N-acetylcysteamine thioester of (4S,5R)-5-hydroxy-4-methyl-2-heptenbic acid using the stereochemically controlled aldol condensation product of N-propionyl-(4S)-4-benzyl-2-oxazolidinone as the starting material: 
This imide was converted to the corresponding aldehyde, and extended at the carbonyl group by a Wittig reaction to obtain the desired triketide as the ethyl ester which was then hydrolyzed and converted to the acylcysteamine thioester in a two-step process. Yields were improved by addition of steps protecting the alcohol, Cane, D. E., et al., J Am Chem Soc (1993) 115:527-535. However, this approach clearly does not lend itself to efficient modular solid-phase synthesis since the building of the triketide chain is nonlinearxe2x80x94i.e., the condensations and the Wittig reactions extend the diketide in opposing directions. Each new analogrequires complete passage through the synthesis with no common intermediates.
While it is clear that thioester forms of acyl moieties, diketides and triketides can be incorporated by PKS systems, to date, little has been reported concerning the optimal thioesters to produce the desired polyketides other than that N-acetylcysteamine thioesters are generally effective as compared with the free carboxylic acids or their oxy-esters (Cane and Yan). It may be expected, however, that the nature of the thioester, e.g. the acyl group in an N-acylcysteamine thioester, might influence such important factors as water solubility, transport into the bacterial cell, metabolism, and recognition by the PKS. A synthetic method for producing variation in the thioester group itself would thus be advantageous.
The present invention offers both improved efficiency in the synthesis of optically-pure diketide thioester intermediates and an approach which provides for efficient extension of the diketides into the corresponding triketide thioesters and provides for additional condensation steps to extend the oligoketide still further. The present invention further provides a method: for synthesis of racemic rather than optically pure diketide thioesters. The racemic materials constitute low-cost alternatives for large-scale production of novel polyketides by fermentation.
The invention offers improvements in the synthesis of oligoketide thioester intermediates. These intermediates can then be incorporated into pathways for the synthesis of novel polyketides using native or modified polyketide synthase (PKS) systems. The invention offers an improvement in the efficiency of diketide synthesis as well as a method for synthesis of triketides and oligoketides in general which is adapted to efficient, linear, solid-phase synthesis. The invention further provides a method to produce racemic diketide thioesters in an economical manner. The invention further provides a method for producing novel polyketides suitable for further modification through the introduction of unique functionalities.
Thus, in one aspect, the invention is directed to the conversion of an acyl imide such as that of a diketide, triketide or oligoketide directly to an N-acylcysteamine thioester by treating the imide with a salt of the corresponding mercaptan. For the synthesis of optically active oligoketide thioesters starting from chiral oxazolidinones, this is done in the presence of a Lewis acid to facilitate the reaction and preserve stereochemical purity. For the synthesis of racemic oligoketide thioesters starting from achiral benzoxazolones, the Lewis acid is not required. This method obviates the intermediate steps of imide hydrolysis, alcohol protection, thioesterification, and deprotection used by previous methods. This method is particularly advantageous for solid-phase synthesis, as it allows for generation of the product with simultaneous cleavage of the oligoketide from the solid support. A particularly facile process using transthiolation of thioesters is given.
In a second aspect, the invention is directed to a method to synthesize racemic diketides and their derivatives through the titanium-mediated aldol condensation between N-acyl-2-benzoxazolones with aldehydes, followed by reaction of the aldol products with nucleophiles to yield the desired derivatives. This method provides a direct route to various oligoketide derivatives, including esters and amides, and is particularly advantageous for the multi-kilogram, economical synthesis of diketide N-acylcysteamine thioesters required for fermentation. As the relative chirality of the carbons at positions 2 and 3 of the attached acyl group is preserved, the racemic mixture will contain one isomer which can be utilized by the PKS and only one additional isomer which cannot. This is in contrast to production of the four possible diestereomerswhich would result in utilization of only one-quarter of the available molecules.
Thus, in still another aspect, the invention is directed to methods to synthesize diketides and triketides which can be used to produce macrolides with functional substituents for example at the 13- and 14-positions by employing, for example, alkenyl- or benzyloxy-aldehydes to introduce starter unit and/or first extender moiety equivalents containing derivatizable groups. The benzyloxy group can readily be converted to a hydroxyl by reduction and then mesylated to provide a suitable leaving group for replacement with nucleophiles, including halides, azides, amines, thiols, other alcohols, and cyanide. The alkenyl group can be.functionalized by any of numerous methods known in the art, including Heck coupling to introduce aryl groups. Such derivatizations can be performed either on the oligoketide or on the polyketides which are produced upon feeding of the oligoketides to suitable PKS systems or cultures of microorganisms.
In an additional aspect, the invention is directed to methods to synthesize oligoketide thioesters using solid-phase combinatorial chemistry. These methods are particularly advantageous when a library of oligoketide thioesters is desired.
In summary, because the invention permits a wide variety of diketide and triketide thioesters to be synthesized in a facile and economic manner, it is possible to prepare a wide variety of polyketides and their tailored derivatives taking advantage of the availability of both recombinant and natively produced polyketide synthase systems and tailoring enzymes, as well as employing chemical transformations using side-chain functional groups.
In still other aspects, the invention relates to feeding diketides or triketides, prepared by the methods of the invention, to suitable PKS systems in vitro or in vivo to obtain oligoketides or polyketides and further converting said polyketides to antibiotics by glycosylation and/or other modifications. The invention also relates to novel intermediates and the resulting modified polyketides and antibiotics.
FIG. 1 sets forth structures of illustrative suitable N-acyl cysteamines.
FIG. 2 illustrates the method for conversion of N-acyloxazolidinones into N-acylcysteamine thioesters.
FIG. 3 illustrates methods for the conversion of N-acyl-2-benzoxazolones into various acyl derivatives, including N-acylcysteamine thioesters.
FIG. 4 illustrates the transthioesterification method developed for use with the diketide benzoxazolones.
FIG. 5 illustrates the formation of N-acyl-2-benzoxazolone and the aldol condensation between N-acyl-2-benzoxazolones and aldehydes, used to prepare intermediates for the synthesis of racemic diketides.
FIG. 6 illustrates the rationale for enforcement of syn-stereochemistry by the benzoxazolone auxiliary.
FIG. 7 illustrates typical diketides, shown as their N-acylcysteamine (SNAC) thioesters, prepared according to the invention.
FIG. 8 illustrates synthesis of oligoketide thioesters using solid-phase chemistry.